Anti-Bed Bug Monoclonal Antibodies and Methods of Making and Uses Thereof

ABSTRACT

The present disclosure provides anti-bed bug monoclonal antibodies and antigen-binding fragments thereof as well as compositions and kits comprising the same. The present disclosure also provides methods of making monoclonal antibodies and antigen-binding fragments thereof and methods of using the same to detect bed bugs.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of the filing date of U.S. Appl. No.62/244,189, filed Oct. 21, 2015, which is hereby incorporated byreference in its entirety.

FIELD

The present disclosure relates to anti-bed bug monoclonal antibodies,compositions comprising such antibodies, and methods of making and usingsuch antibodies.

BACKGROUND

The scale and the number abed bug infestations in the United States haveincreased for over a decade. Bed bugs are commonly found in multi-unithousing, such as apartments, dormitories, nursing homes, and hotels, andpublic venues such as theaters, public transportation, and shoppingmalls.

While bed bugs are not known vectors of any pathogen, there is someevidence that bed bugs could act as mechanical vectors of Hepatitis Bvirus (Blow et al., J. Med. Entomol. 38: 694-700 (2001)).Methicillin-resistant Staphylococcus aureus (MRSA) andvancomycin-resistant Enterococcus faecium were recovered from bed bugsassociated with a homeless shelter (Lowe et al., Emerg. Infect. Dis. 17:1132-1134 (2011)).

Detection strategies currently in use include:

Visual detection: Personally checking mattresses and bedsprings,upholstery, and carpets for bed bugs, shed exoskeletons, or fecaldroplets takes time and is often complicated by cryptic, inaccessibleharborages;

Canine detection: Results with bed bug-sniffing dogs are highlyvariable. Success depends on the dog and trainer and type of entrainmentand reward. Canine detection yields unacceptably high numbers of falsepositives, and its conspicuousness results in unpleasant publicrelations;

Active or passive monitors. Active monitors—such as Verdi® by FMC,CDC3000® by Stern Environmental, or NightWatch® by BioSensory, Inc. relyon custom pheromone blends or carbon dioxide, to attract bed bugs to atrap. Passive monitors, such as the ClimbUp® Insect interceptor, areplaced near a sleeping person and use the heat and carbon dioxide ofthat person to attract and trap bed bugs. The efficacy of active andpassive monitors depends on bed bug population density and may miss orunderestimate small introductions of bugs. Most monitors also have anunacceptably large footprint, require specially trained personnel, andcost too much, in addition, both strategies involve actually handlingdead bugs, which most people find unpleasant;

Other approaches to bed bug detection include:

Multiplex polymerase chain reaction (PCR) to distinguish bed bug eggs orbug fragments from human dwellings. This technique depends on physicallyrecovering eggs or bug fragments and processing them using standardmolecular biology reagents and techniques (Szalanski et al., J. Med.Entomol. 48: 937-940 (2011));

Microextraction of air samples to identify two well-characterizedvolatile pheromones, (E)-2-hexenal and (E)-2-oetenal, by gaschromatography and mass spectrometry (Eom et al., Anal Chim Acta 716:2-10 (2012));

The use of infrared sensors, microphones, and a piezoelectric sensor todetect locomotion (Mankin et al., J. Econ. Entomol. 103: 1636-1646(2010));

The detection of nitrophorin, a bed bug-specific salivary antigen (U.S.Pat. No. 7,743,552);

The detection of human blood antigens in bed bug excreta (U.S. Pat. No.8,460,890): and

The detection of bed bug antigens by polyclonal antibodies (U.S.Publication No. 2015/0064727).

The number of available detection options is limited and there is a needfor a convenient, easy-to-use detection method that rapidly,reproducibly, effectively, and directly detects bed hugs. In contrast tothe prior approaches, the present invention provides monoclonalantibodies that satisfy the need.

SUMMARY

The present invention is directed to an antibody produced by thehybridoma deposited at the American Type Culture Collection (ATCC) underAccession Number PTA-122644 [BB2], or an antigen-binding fragmentthereof.

The present invention is directed to an antibody produced by thehybridoma deposited at the ATCC under Accession Number PTA-112645 [BB7],or an antigen-binding fragment thereof.

The present invention is directed to a monoclonal antibody or anantigen-binding fragment thereof comprising the heavy chain and lightchain complementarity determining regions (CDRs) of an antibody producedby the hybridoma deposited at the ATCC under Accession Number PTA-122644[BB2] or an antibody produced by the hybridoma deposited at the ATCCunder Accession Number PTA-122645 [BB7]. In certain embodiments, theantibody or antigen-binding fragment thereof comprises the heavy andlight chain variable regions of the antibody produced by the hybridomadeposited at the ATCC under Accession Number PTA-122644 [BB2] or theantibody produced by the hybridoma deposited at the ATCC under AccessionNumber PTA-122645 [BB7]. In certain embodiments, the antibody orantigen-binding fragment thereof comprises the heavy and light chains ofthe antibody produced by the hybridoma deposited at the ATCC underAccession Number PTA-122644 [BB2] or the antibody produced by thehybridoma deposited at the ATCC under Accession Number PTA-122645 [BB7].

In certain embodiments, the antibody or antigen-binding fragment of anyof the above inventions and embodiments is capable of binding to a bedbug antigen in a lysate of whole bed bugs or an extract of collectionpaper comprising bed hug waste material.

The present invention is directed to a mutant of an antibody produced bythe hybridoma deposited at the ATCC under Accession Number PTA-122644[BB2] or a mutant of an antibody produced by the bybridoma deposited atthe ATCC under Accession Number PTA-122645 [BB7], wherein the mutant iscapable of binding to a bed bug antigen in a lysate of whole bed bugs oran extract of collection paper comprising bed bug waste material.

The present invention is directed to a conjugated monoclonal antibody ora conjugated antigen binding fragment comprising any of the antibodies,antigen-binding fragments, or mutants of the above inventions orembodiments and a detection agent. In certain embodiments, the detectionagent is colloidal gold. In certain embodiments, the conjugated antibodyor conjugated antigen-binding fragment comprises an antibody produced bythe hybridoma deposited at the ATCC under Accession Number PTA-122644[BB2], or an antigen-binding fragment thereof, or an antibody producedby the hybridoma deposited at the ATCC under Accession Number PTA-122645[BB7], or an antigen-binding fragment thereof.

The present invention is directed to a composition comprising any of theantibodies, antigen-binding fragments, mutants, or conjugated antibodiesor conjugated antigen-binding fragments of the above inventions orembodiments, or a combination thereof. In certain embodiments, thecomposition comprises an antibody produced by the hybridoma deposited atthe ATCC under Accession Number PTA-122644 [BB2] and an antibodyproduced by the hybridoma deposited at the ATCC under Accession NumberPTA-122645 [BB7]. In certain embodiments, the composition comprises anantibody produced by the hybridoma deposited at the ATCC under AccessionNumber PTA-122644 [BB2], and a conjugated antibody comprising theantibody produced by the hybridoma deposited at the ATCC. underAccession Number PTA-122645 [BB7] and a detection agent. In certainembodiments, the composition comprises an antibody produced by thehybridoma deposited at the ATCC under Accession Number PTA-122645 [BB7],and a conjugated antibody comprising the antibody produced by thehybridoma deposited at the ATCC under Accession Number PTA-122644 [BB2]and a detection agent.

The present invention is directed to a kit comprising any of the aboveinventions or embodiments, or a combination thereof.

The present invention is directed to a hybridoma capable of producing anantibody, wherein the hybridoma is deposited at the ATCC under AccessionNumber PTA 122644 [BB2] or wherein the hybridoma is deposited at theATCC under Accession Number PTA-122645 [BB7].

The present invention is directed to an isolated cell producing anantibody, antigen-binding fragment, or mutant of any of the aboveinventions or embodiments.

The present invention is directed to a method of making an antibody,antigen-binding fragment, or mutant of any of the above inventions orembodiments, comprising culturing an isolated cell producing theantibody, antigen-binding fragment, or mutant, and isolating theantibody, antigen-binding fragment, or mutant from the cultured cell.

The present invention is directed to a method of detecting bed bugs,comprising contacting a sample comprising a bed bug antigen with any ofthe antibodies, antigen-binding fragments, mutants, conjugatedantibodies or conjugated antigen-binding fragments, or compositions ofthe above inventions or embodiments, or a combination thereof, anddetecting binding of the bed bug antigen to the antibody orantigen-binding fragment, mutant, conjugated antibody or conjugatedantigen-binding fragment, composition, or combination thereof. Incertain embodiments, the sample is contacted with an antibody of any ofthe above inventions or embodiments and a conjugated antibody of any ofthe above inventions or embodiments, in certain embodiments, theantibody is produced by the hybridoma deposited at the ATCC underAccession Number PTA-122644 [BB2], and the conjugated antibody comprisesthe antibody produced by the hybridoma deposited at the ATCC underAccession Number PTA-122645 [BB7] and a detection agent. In certainembodiments, the antibody is produced by the hybridoma deposited at theATCC under Accession Number PTA-122645 [BB7], and the conjugatedantibody comprises the antibody produced by the hybridoma deposited atthe ATCC under Accession Number PTA-122644 [BB2] and a detection agent,in certain embodiments, the detecting comprises performing a lateralflow assay. In certain embodiments, the method further comprisescollecting a sample comprising the bed bug antigen with a collectiondevice and extracting antigens from the sample. In certain embodiments,the collection device is a swab.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of a sandwich capture assay using the BB2 andBB7 anti-bed bug monoclonal antibodies, Nitrocellulose strips are shownwith binding of bed bug antigen indicated by dots associated with thepresence of gold-conjugated BB2 or BB7 monoclonal antibody (“DetectorAb”), with the pH of the gold-conjugated antibody as indicated. Teststrips are indicated by “bed bug” for the antigen. Negative controlstrips are indicated by “none” for the antigen, with PBS added to thestrips instead of bed bug antigen. The capture antibodies associatedwith lanes 1-3 were rabbit polyclonal anti-bed bug antibody (lane 1),the BB2 monoclonal antibody (lane 2), and the BB7 monoclonal antibody(lane 3).

FIGS. 2-7 show the results of a lateral flow immunoassay for swabsamples obtained from the noted levels of bed bug infestation, withlevel 0 containing no bed bugs and level 8 containing the highest levelof bed bugs. Swab samples were extracted in extraction butler Icontaining 1× Tris-HCl (pH 7.6), 0.05% NaN₃, 0.1% BSA, and 0.1% Tween-20and the noted dilutions were applied to test strips. “Buffer” indicatesa negative control test strip in which buffer I was applied instead of aswab sample. All of the strips contain a positive control stripe of goatanti-mouse antibody, which is located above a BB7 capture antibodystripe for binding bed bug antigen. The stripes become visuallydetectable only upon binding of the gold-conjugated mouse monoclonal BB2antibody to the goat anti-mouse positive control stripe or toimmobilized bed bug antigen captured by the BB7 stripe. The line on the“Buffer” strip and the corresponding lines on the test strips are thepositive controls that indicate binding of the goat anti mouse antibodyto the gold-conjugated BB2 detector antibody. Only positive controls aredetectable in FIG. 2 due to the absence of antigen. The lines beneaththe positive control lines in FIGS. 3-7 indicate binding of thegold-conjugated BB2 antibody to immobilized bed bug antigen captured bythe 1387 stripe. FIGS. 6-7 show noticeable dirt and residue for lessdilute samples at the bottom of the test strips associated with thehigher levels of bed bug infestation.

FIGS. 8A and 8B are graphs based on measurements made by the Axxin teststrip reader of swab samples for levels 2, 4, and 8 extracted inbuffer 1. In FIG. 8A, the x-axis “concentration” is the dilutionassociated with the measured swab samples, and the y-axis is the valuefor the “test line area” provided by the Axxin test strip reader. InFIG. 8B, the value obtained for buffer 1 as a negative control (i.e.,“Bo”) was divided by itself to yield a normalized value of 1. Thenegative control reading (Bo) was then divided by the test line area 03)for each test sample dilution in the level, where smaller values under 1suggest larger amounts of bed bug antigen and values above 1 indicateabsence of bed bug antigen.

FIGS. 9-11 show the results of a lateral flow immunoassay for swabsamples obtained from the noted levels of bed bug infestation. Swabsamples were extracted in extraction buffer 2 containing 1× Tris-HCl (pH7.6), 0.05% NaN₃, 0.1% BSA, and 0.2% Tween-20 and the noted dilutionswere applied to test strips. FIG. 11 shows noticeable dirt and residuefor less dilute samples at the bottom of the test strips associated withthe higher levels of bed bug infestation. Labeling of the strips andinterpretation of results is as described for FIGS. 2-7.

FIGS. 12A and 12B are graphs based on measurements made by the Axxintest strip reader of swab samples for levels 4, 5, and 8 extracted inbuffer 2. Labeling of the graphs is as described for FIGS. 8A and 8B.

FIGS. 13-17 show the results of a lateral flow immunoassay for swabsamples obtained from the noted levels of bed bug infestation. Swabsamples were extracted in extraction buffer 3 containing 1× Tris-HCl (pH7.6), 0.05% NaN₃, 0.25% BSA, and 0.1% Tween-20 and the noted dilutionswere applied to test strips. FIGS. 16-17 show noticeable dirt andresidue for less dilute samples at the bottom of the test stripsassociated with the higher levels of bed bug infestation. Labeling ofthe strips and interpretation of results is as described for FIGS. 2-7.

FIGS. 18A and 18B are graphs based on measurements made by the Axxintest strip reader of swab samples for levels 2, 5, and 8 extracted inbuffer 3. Labeling of the graphs is as described for FIGS. 8A and 8B.

FIGS. 19A and 19B are graphs based on measurements made by the Axxintest strip reader showing a comparison of results for level 8 swabsamples extracted in buffers 1, 2, and 3. Labeling of the graphs is asdescribed for FIGS. 8A and 8B.

FIG. 20 is a graph based on measurements made by the Axxin test stripreader for nine replicates of extraction buffer 1 as a negative controland nine replicates each of 1/2048, 1/512, and 1/128 dilutions for level7 swab samples extracted in buffer 1. The x-axis indicates the ninereplicates by “trial” number; the y-axis is as described for FIG. 8A.

DETAILED DESCRIPTION

Provided herein are monoclonal antibodies and antigen-binding fragmentsthereof that hind to a bed hug antigen, compositions and kits comprisingthe antibodies and antigen-binding fragments, hybridomas, polypeptides,polynucleotides, vectors, cells capable of producing the antibodies andantigen-binding fragments, methods of making the antibodies andantigen-binding fragments, and methods of using the antibodies andantigen-binding fragments, for example, in the detection of bed bugs.

Terminology

As used herein, the term “bed bug” refers to any Cimex species or strainthereof.

The terms “antibody” and “antibodies” are terms of art and can be usedinterchangeably herein to refer to a molecule or molecules with anantigen-binding site that specifically binds an antigen.

The term “monoclonal antibody” refers to a homogeneous antibodypopulation involved in the specific recognition and binding of a singleantigenic determinant, or epitope. This is in contrast to polyclonalantibodies that typically include different antibodies directed againstdifferent antigenic determinants. Furthermore, “monoclonal antibody”refers to such antibodies made in any number of manners, including, butnot limited to, by hybridoma, phage selection, recombinant expression,and transgenic animals.

The term “antigen-binding fragment” refers to a portion of an antibodythat is capable of specifically binding to an antigen. Examples ofantibody fragments include, but are not limited to heavy chain variableregion fragments, light chain variable region fragments, Fab, Fab′,F(ab′)2, scFv fragments, Fv fragments, linear antibodies, single chainantibodies, multispecific antibodies, minibodies diabodies, triabodies,and tetrabodies.

The terms “variable region” or “variable domain” are terms of art andcan be us d interchangeably herein to refer to a portion of an antibodythat differs extensively in sequence among antibodies and is used in thebinding and specificity of a particular antibody for its particularantigen. The variable regions of the heavy and light chain each consistof four framework regions (FR) connected by three complementaritydetermining regions (CDRs) also known as hypervatiable regions. The CDRsin each chain contribute to the formation of the antigen-binding site ofantibodies.

The term “specifically binds” refers to molecules that bind to anantigen (e.g., epitope or immune complex) as such binding is understoodby one skilled in the art. For example, a molecule that specificallybinds to an antigen can bind to other peptides or polypeptides,generally with lower affinity as determined by, e.g., immunoassays,BIAcore, KinExA 3000 instrument (Sapidyne Instruments, Boise, Id.), orother assays known in the art.

As used herein, the term “detecting” encompasses quantitative andqualitative detection.

As used herein, the term “effective amount” refers to the amount of thatachieves a desired effect.

As used herein, the terms “host cell” and “cell” can be usedinterchangeably and can refer to any type of cell, e.g., a primary cell,a cell in culture, or a cell from a cell line, including a hybridoma.

An antibody, antigen-binding fragment, host cell, and cell as referredto herein includes “isolated” forms that have been separated orrecovered from a component of their native environment, such asseparation or removal from contaminants that would interfere with usesof the antibody, antigen-binding fragment, host cell, or cell, in whichsuch contaminants may include enzymes, hormones, and other proteinaceousor nonproteinaceotis materials.

Monoclonal Antibodies, Compositions, Kits, Hybridomas, Polypeptides,Polynucleotides, Vectors, Cells, and Methods of Making

The present invention is directed to an antibody produced by thehybridoma deposited at the American Type Culture Collection (ATCC) underAccession Number PTA-122644, or an antigen-binding fragment thereof. Theanti-bed bug monoclonal antibody designated herein as BB2 is produced bythe hybridoma deposited at the ATCC under Accession Number PTA-122644.

The present invention is directed to an antibody produced by thehybridoma deposited at the ATCC under Accession Number PTA-122645, or anantigen-binding fragment thereof. The anti-bed bug monoclonal antibodydesignated herein as BB7 is produced by the hybridoma deposited at theATCC under Accession Number PTA-122645.

The present invention is directed to a monoclonal antibody or an antigenbinding fragment thereof comprising the heavy chain and light chaincomplementarity determining regions (CDRs) of the antibody produced bythe hybridoma deposited at the ATCC under Accession Number PTA-122644[BB2] or the antibody produced by the hybridoma deposited at the ATCCunder Accession Number PTA-122645 [BB7] (see e.g., the discussion ofCDRs in Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983), and Chothia etal., J. Mol. Biol. 196:901-917 (1987)). Methods for determining CDRs arewell-known, including an approach based on cross-species sequencevariability (i.e., Kabat et al. Sequences of Proteins of ImmunologicalInterest, (5th ed., 1991, National Institutes of Health, Bethesda,Md.)), and an approach based on crystallographic studies ofantigen-antibody complexes (Al-lazikani et al., J. Molec. Biol.273:927-948 (1997)). In addition, combinations of these two approachescan be used to determine CDRs. CDRs also can be determined according toLefranc M-P, The Immunologist 7: 132-136 (1999); Lefranc M-P, et al.,Nucleic Acid Res 27: 209-212 (1999); MacCallum R M et al., J. Mol. Biol.262: 732-745 (1996); Martin A. “Protein Sequence and Structure Analysisof Antibody Variable Domains,” in Antibody Engineering, Kontemiann andDübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001);and Oxford Molecular's AbM antibody modeling software (Oxford MolecularGroup, Inc.).

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises the heavy and light chain variable regions of the antibodyproduced by the hybridoma deposited at the ATCC under Accession NumberPTA-122644 [BB2] or the antibody produced by the hybridoma deposited atthe ATCC under Accession Number PTA-122645 [BB7]. Generally, a variableregion is located at about the amino-terminal 110 to 120 amino acids inthe mature heavy chain and about the amino-terminal 90 to 115 aminoacids in the mature light chain.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises the hear and light chains of the antibody produced by thehybridoma deposited at the ATCC under Accession Number PTA -122644 [BB2]or the antibody produced by the hybridoma deposited at the ATCC underAccession Number PTA-122645 [BB7]. Each heavy chain comprises a heavychain variable region and a heavy chain constant region. The heavy chainconstant region comprises three domains, CH1, CH2 and CH3. Each lightchain comprises a light chain variable region and a light chain constantregion. The light chain constant region comprises one domain (CL1).

The monoclonal antibodies of the invention can include, but are notlimited to, recombinantly produced antibodies, human antibodies,humanized antibodies, chimeric antibodies, multispecific antibodies suchas bispecific antibodies, fusion proteins comprising an antigendetermination portion of an antibody, and any other modifiedimmunoglobulin molecule comprising an antigen recognition site so longas the antibodies exhibit the desired activity. Antibodies can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG₁,IgG₂, IgG₃, IgG₄, IgA₁, or IgA₂), or any subclass (e.g., IgG_(2a) orIgG_(2b)) of immunoglobulin molecule. Techniques for the production ofantibodies will be apparent to the skilled practitioner.

Monoclonal antibodies can be prepared, for example, using hybridomamethods, such as those described by Kohler and Milstein, Nature 256:495(1975). Using the h hybridoma method, a mouse, hamster, or otherappropriate host animal, is immunized to elicit the production bylymphocytes of antibodies that will specifically bind to an immunizingantigen. Lymphocytes can also be immunized in vitro. Followingimmunization, the lymphocytes are isolated and fused with a suitablemyeloma cell line using, for example, polyethylene glycol, to formhybridoma cells that can then be selected away from unfused lymphocytesand myeloma cells.

Monoclonal antibodies can also be made using recombinant DNA methods asdescribed in U.S. Pat. No. 4,816,567. The polynucleotides encoding amonoclonal antibody are isolated from mature B-cells or hybridoma cell,such as by RT-PCR using oligonucleotide primers that specificallyamplify the genes encoding the heavy and light chains of the antibody,and their sequence is determined using conventional procedures. Theisolated polynucleotides encoding the heavy and light chains are thencloned into suitable expression vectors, which allow for generation ofmonoclonal antibodies when transfected into host cells, including, butnot limited to, E. coli cells, simian COS cells, Chinese hamster ovary(CHO) cells, or myeloma cells. Also, recombinant monoclonal antibodiesor fragments thereof of the desired species can be isolated from phagedisplay libraries expressing CDRs of the desired species (see, e.g.,McCafferty et al., Nature 348:552-554 (1990); Clackson et al., Nature352:624-628 (1991); and Marks et al., J. Mol. Biol. 222:581-597 (1991)).

Antigen-binding fragments of the invention can be produced by any knownmethod and include a portion of an antibody that is capable ofspecifically binding to an antigen. Examples of antibody fragmentsinclude, but are not limited to, heavy chain variable region fragments,light chain variable region fragments, Fab, Fab′, F(ab′)2, scFvfragments, Fv fragments, linear antibodies, single chain antibodies,multispecific antibodies, minibodies, diabodies, triabodies, andtetrabodies see, e.g., Hudson and Souriau, Nature Med. 9: 129-134 (2003)and U.S. Pat. No. 5,641,870). Traditionally, these fragments are derivedvia proteolytic digestion of intact antibodies (see, e.g., Morimoto etal., Journal of Biochemical and Biophysical Methods 24:107-117 (1993);Brennan et a1, Science 229:81 (1985)). In certain embodiments, antibodyfragments are produced recombinantly. For example, antibody fragmentscan be expressed in and secreted from E. coli or other host cells, thusallowing the production of large amounts of the fragments. Such antibodyfragments can also be isolated from the antibody phage libraries. Othertechniques for the production of antibody fragments will be apparent tothe skilled practitioner.

In certain embodiments, any of the above-mentioned antibodies orantigen-binding fragments thereof i capable of binding to a bed bugantigen in a lysate of whole bed bugs or an extract of collection papercomprising bed bug waste material. The methods for producing lysates ofwhole bed bugs and extracts of collection paper comprising bed bug wastematerial will be apparent to the skilled practitioner based on knownextraction techniques and the methods disclosed in Example 1. Whole bedbugs include nymphs, males, and/or females can be obtained from an areaof infestation or an experimentally or commercially maintained bed bugcolony, and can include any Cimex species or strain, including, but notlimited to, Cimex lectularius and the Harlan strain. Collection papercomprising bed bug waste material can include bed “bug excreta and/ortissues, for example, and can be obtained from commercial sources asdiscussed in Example 1.

The present invention is also directed to a mutant of the antibodyproduced by the hybridoma deposited at the ATCC under Accession NumberPTA-122644 [BB2] or the antibody produced by the hybridoma deposited atthe ATCC under Accession Number PTA-122645 [BB7]. Mutants can contain,for example, conservative substitution mutations, i.e. the substitutionof one or more amino acids by similar amino acids. For example,conservative substitution refers to the substitution of an amino acidwith another within the same general class such as, for example, oneacidic amino acid with another acidic amino acid, one basic amino acidwith another basic amino acid or one neutral amino acid by anotherneutral amino acid. What is intended by a conservative amino acidsubstitution is well known in the art. Mutations can also includedeletions, insertions, inversions, and repeats. Mutations can beintroduced by general molecular biology methods known in the artincluding, but not limited to, error-prone PCR, oligonucleotide-directedmutagenesis, site-directed mutagenesis, and heavy or light chainshuffling.

The present invention is directed to a monoclonal antibody orantigen-binding fragment thereof having one or more characteristics thatare substantially similar to those of the antibody produced by thehybridoma deposited at the ATCC under Accession Number PTA-122644 [BB2]or the antibody produced by the hybridoma deposited at the ATCC underAccession Number PTA-122645 [BB7]. The phrase “substantially similar” asused herein denotes a sufficiently high degree of similarity between twocharacteristics such that one of skill in the art would consider thedifference to be of little or no biological and/or statisticalsignificance. In certain embodiments, the difference between twonumerical values can be less than about 15%, 10%. 5%, 2%, or 1%. Thecharacteristics of the deposited antibodies can include one or moreproperties, such as, but not limited to, binding specificity

Kd value), antigenic determinants/epitope, and polynucleotide orpolypeptide sequences. In certain embodiments, the monoclonal antibodyor antigen-binding fragment thereof has a polynucleotide or polypeptidesequence that is at least 90%-99%, at least 95%-99%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the polynucleotide or polypeptide sequence of the antibodyproduced by the hybridoma deposited at the ATCC under Accession NumberPTA-122644 [BB2] or the antibody produced by the hybridoma deposited atthe,ATCC under Accession Number PTA-122645 [BB7]. In certainembodiments, the monoclonal antibody or an ti,gen-binding fragmentthereof has one or more of the same characteristics as the antibodyproduced by the hybridoma deposited at the ATCC under Accession NumberPTA-122644 [BB2] or the antibody produced by the hy“bridoma deposited atthe ATCC under Accession Number PTA-122645 [BB7]. In certainembodiments, the monoclonal antibody or antigen-binding fragment thereofbinds to the same antigenic determinant/epitope as the antibody producedby the hybridoma deposited at the ATCC under Accession Number PTA-122644[BB2] or the antibody produced by the hybridoma deposited at the ATCCunder Accession Number PTA-122645 [BB7].

The term “epitope” or “antigenic determinant” can be usedinterchangeably herein to refer to that portion of an antigen capable ofbeing recognized and specifically bound by a particular antibody. Anepitope typically includes at least 3, and more usually, at least 5 or8-10 amino acids in a unique spatial conformation. An epitope can be,for example, contiguous amino acids of a polypeptide (linear orcontiguous epitope) or an epitope can, for example, come together fromtwo or more non-contiguous regions of a polypeptide or polypeptides(conformational, non-linear, discontinuous, or non-contiguous epitope).The epitope to which an antibody binds can be determined by, e.g., NMRspectroscopy, X-ray diffraction crystallography studies, ELISA assays,hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquidchromatography electrospray mass spectrometry), array-basedoligo-peptide scanning assays, and/or mutagenesis mapping (e.g.,site-directed mutagenesis mapping). For X-ray crystallography,crystallization may be, accomplished using any of the known methods inthe art (e.g., Giegé R et al., (1994) Acta Crystallogr D BiolCrystallogr 50(Pt 4): 339-350; McPherson A (1990) Fur l Biochem 189:1-23; Chayen N E (1997) Structure 5: 1269-1274; McPherson A (1976) jBiol Chem 251: 6300-6303). Antibody:antigen crystals can be studiedusing well known X-ray diffraction techniques and can be refined usingcomputer software such as X-PLOR (Yale University, 1992, distributed byMolecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114& 115, eds Wyckoff H W et al.; U.S. 2004/0014194), and BUSTER (BricogneG (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G(1997) Meth Enzymol 276A: 361-423, ed Carter C W; Roversi P et at.,(2000) Acta Ctystallogr D Biol Crystallogr 56(Pt 10): 1316-1323),Mutagenesis mapping studies can be accomplished using any method knownto one of skill in the art. See, e.g., Champe M al., (1995) J Biol Chem270; 1388-1394 and Cunningham B C & Wells J A (1989) Science 244:1081-1085 for a description of mutagenesis techniques, including alaninescanning mutagenesis techniques.

The present invention is directed to a conjugated monoclonal antibody orconjugated antigen-binding fragment comprising any of the antibodies,antigen binding fragments, or mutants of the invention and a detectionagent The detection agent can be conjugated directly or indirectly tothe antibody, antigen-binding fragment, or mutant. The detection agentcan be detectable by itself or, in the case of an enzymatic label, cancatalyze chemical alteration of a substrate compound or compositionwhich is detectable. The detection agent includes, but is not limitedto, a radiolabel, a fluorophore, a chromophore, an imaging agent, or ametal, including a metal ion. In certain embodiments, the detectionagent is colloidal gold or gold nanoparticles. In certain embodiments,the colloidal gold or gold nanoparticies is comprised of gold particleshaving a size of 1-300 nm, 1-250 nm, 10-200 nm, 20-150 nm, 20-100 nm,20-80 nm, 20-60 nm 20-50 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm,80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, or 300 nm. In certainembodiments, the conjugated antibody or conjugated antigen-bindingfragment comprises the antibody produced by the hybridoma deposited atthe ATCC under Accession Number PTA-122641 [BB2], or an antigen-bindingfragment thereof, or the antibody produced by the hybridoma deposited atthe ATCC under Accession Number PTA-122645 [BB7], or an antigen-bindingfragment thereof.

The present invention is directed to a composition comprising any of theantibodies or antigen binding fragments thereof, mutants, or conjugatedantibodies or conjugated antigen-binding fragments of the invention, ora combination thereof In certain embodiments, the composition comprisesthe antibody produced by the hybridoma deposited at the ATCC underAccession Number PTA-122644 [BB2] and the antibody produced by thehybridoma deposited at the ATCC under Accession Number PTA-122645 [BB7].In certain embodiments, the composition comprises the antibody producedby the hybridoma deposited at the ATCC under Accession Number PTA-122644[BB2], and a conjugated antibody comprising the antibody produced by thehybridoma deposited at the ATCC under Accession Number PTA-122645 [BB7]and a detection agent. In certain embodiments, the composition comprisesthe antibody produced by the hybridoma deposited at the ATCC underAccession Number PTA-122645 [BB7], and a conjugated antibody comprisingthe antibody produced by the hybridoma deposited at the ATCC underAccession Number PTA-122644 [BB2] and a detection agent.

The present invention is directed to a kit comprising any of theantibodies or antigen binding fragments thereof, mutants, conjugatedantibodies or conjugated antigen-binding fragments, or compositions ofthe invention, or a combination thereof In certain embodiments, a kitcomprises at least one component in one or more containers. In someembodiments, the kit comprises components necessary and/or sufficient toperform. a detection assay, including controls, directions forperforming assays, any necessary device, and/or software for analysisand presentation of results. Suitable devices include those disclosed inU.S. Application No. 62/244,188 filed Oct. 21, 2015, titled “Bed BugsDetection Device,” and the U.S. non-provisional application andinternational PCT application thereof filed Oct. 21, 2016, which areincorporated by reference herein in their entireties, as well as U.S.Pat. Nos. 7,220,597 and 7,214,542, both of which are incorporated byreference herein in their entireties.

The present invention is directed to a hybridoma capable of producing anantibody, wherein the hybridoma is deposited at the ATCC under AccessionNumber PTA-122644 [BB2] or wherein the hybridoma is deposited at theATCC under Accession Number PTA-122645 [BB7].

The present invention is directed to an isolated polypeptide comprisingan amino acid sequence at least 90%-99%, at least 95%-99%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98% or at least 99%identical to an amino acid sequence of a heavy or light chain variableregion, or a heavy or light chain, of the antibody produced by thehybridoma deposited at the ATCC under Accession Number PTA-122644 [BB2]or the antibody produced by the hybridoma deposited at the ATCC underAccession Number PTA-122645 [BB7]. In certain embodiments, thepolypeptide comprises the amino acid sequences of the CDRs of a heavy orlight chain variable region of the antibody produced by the hybridomadeposited at the ATCC under Accession Number PTA-122644 [BB2] or theantibody produced by the hybridoma deposited at the ATCC under AccessionNumber PTA-122645 [BB7]. In certain embodiments, the polypeptidecomprises the amino acid sequences of the heavy or light chain variableregion, or heavy or light chain, of the antibody produced by thehybridoma deposited at the ATCC under Accession Number PTA-122644 [BB2]or the antibody produced by the hybridoma deposited. at the ATCC underAccession Number PTA-122645 [BB7].

The present invention is directed to an isolated polynucleotidecomprising a nucleic acid sequence at least 90%-99%, at least 95%-99%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98% orat least 99% identical to a nucleic acid sequence encoding a heavy orlight chain variable region, or a heavy or light chain, of the antibodyproduced by the hybridoma deposited at the ATCC under Accession NumberPTA-122644 [BB2] or the antibody produced by the hybridoma deposited atthe ATCC under Accession Number PTA-122645 [BB7]. In certainembodiments, the polynucleotide comprises nucleic acid sequencesencoding the CDRs of a heavy or light chain variable region of theantibody produced by the hybridoma deposited at the ATCC under AccessionNumber PTA-122644 [BB2] or the antibody produced by the hybridomadeposited at the ATCC under Accession Number PTA-122645 [BB7]. Incertain embodiments, the polynucleotide comprises a nucleic acidsequence encoding the heavy or light chain variable region, or heavy orlight chain, of the antibody produced by the hybridoma deposited at theATCC under Accession Number PTA-122644 [BB2] or the antibody produced bythe hybridoma deposited at the ATCC under Accession Number PTA-122645[BB7].

The present invention is directed to a vector comprising one or more ofthe isolated polynucleotides of the invention. In certain embodiments,the vector is an expression vector.

The present invention is directed to an isolated cell producing anantibody, antigen-binding fragment, or mutant of the invention. Incertain embodiments, the cell is a hybridoma. In certain embodiments,the cell comprises one or more vectors of the invention, In certainembodiments, the cell comprises one or more polynucleotides of theinvention.

The present invention is directed to a method of making an antibody,antigen-binding fragment, or mutant of the invention, comprisingculturing an isolated cell producing the antibody, antigen-bindingfragment, or mutant, and isolating the antibody, antigen-bindingfragment, or mutant from the cultured cell.

Cells include, but are not limited to, hybridomas, prokaryotes, yeast,insect, or higher eukaryotic cells. Hybridomas that produce monoclonalantibodies can be propagated either in vitro culture using standardmethods (coding, Monoclonal Antibodies: Principles and Practice,Academic Press, 1986) or in vivo as ascites tumors in an animal.Prokaryotes include gram negative or gram positive organisms, forexample E. coli or bacilli. Higher eukaryotic cells include, but are notlimited to, established cell lines of mammalian origin. Examples ofsuitable mammalian cell lines include COS-7, L, C127, 3T3, Chinesehamster ovary (CHO), HeLa, and BHK cell lines. In certain embodiments,any of the antibodies, antigen-binding fragments, or mutants of theinvention are produced by isolated cells following transfection of thecells with vectors comprising polynucleotides encoding the sequences ofthe antibodies, antigen-binding fragments, or mutants of the invention.Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are described by Pouwels etal. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985).Mammalian expression vectors can comprise nontranscribed elements suchas an origin of replication, a suitable promoter and enhancer linked tothe gene to be expressed, and other 5′ or 3′ flanking nontranscribedsequences, and 5′ or 3′ nontranslated sequences, such as necessarytibosome binding sites, a polyadenylation site, splice donor andacceptor sites, and transcriptional termination sequences. Baculovirussystems for production of heterologous proteins in insect cells arereviewed by and Luckow and Summers, Bio/Technology 6:47 (1988).

The antibodies, antigen-binding fragments, or mutants of the inventioncan be isolated front the cells or culture medium, or from ascites fluidfor in vivo propagation of hybridomas. Isolation of the antibodies,antigen-binding fragments, or mutants can be according to any suitablemethod. Such standard methods include chromatography (e.g., ionexchange, affinity and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for proteinpurification. Methods known in the art for purifying antibodies andother proteins include, for example, those described in U.S. PatentPublication Nos. 2008/0312425, 2008/0177048, and 2009/0187005.

Methods of Detecting Bed Bugs

The present invention is directed to a method of detecting bed bugs,comprising contacting a sample comprising a bed bug antigen with any ofthe antibodies, antigen-binding fragments, mutants, conjugatedantibodies or conjugated antigen-binding fragments, or compositions ofthe invention, or a combination thereof, and detecting binding of thebed bug antigen to the antibody or antigen-binding fragment, mutant,conjugated antibody or conjugated antigen-binding fragment, composition,or combination thereof. “A sample” includes, but is not limited to,whole bed bugs, bed bugs parts, bed bug waste material, lysates orextracts thereof, extracts of collection paper comprising bed bug wastematerial, and fluids containing the same.

The contacting can be by any suitable method. In certain embodiments,the contacting is by application of a sample comprising the bed bugantigen to an antibody, antigen-binding fragment, mutant, conjugatedantibody or conjugated antigen-binding fragment, or composition of theinvention, or a combination thereof that is immobilized or otherwiselocated on a surface. Any acceptable surface can be used, as will beappreciated by the skilled practitioner, including, but not limited to,a nitrocellulose membrane or a pad composed of a suitable material, andcan include a sandwich, well, or lateral flow design. In certainembodiments, the sample is contacted with an antibody of the inventionand a conjugated antibody of the invention. In certain embodiments, theantibody is produced by the hybridoma deposited at the ATCC underAccession Number PTA-122644 [BB2], and the conjugated antibody comprisesthe antibody produced by the hybridoma deposited at the ATCC underAccession Number PTA-122645 [BB7] and a detection agent. In certainembodiments, the antibody is produced by the hybridoma deposited at theATCC under Accession Number PTA-122645 [BB7], and the conjugatedantibody comprises the antibody produced by the hybr.idoma deposited atthe ATCC under Accession Number PTA-112644 [BB2] and a detection agent.In certain embodiments, the contacting further comprises contacting theantibody, antigen-binding fragment, mutant, conjugated antibody orconjugated antigen-binding fragment, or composition of the invention, ora combination thereof with a control sample for comparison with the testsample.

The detecting can be by any suitable method and can include quantitativeor qualitative detection. Such methods include, but are not limited to,antigen-binding assays that are well known in the art, such as lateralflow assays, radioimmunoassays, ELISA (enzyme linked.immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, fluorescentimmunoassays, protein A. immunoassays, and immunohistochemistry (IHC).The detection can include visual analysis of a colorimetric,fluorescent, or luminescent reaction, for example, or can include use ofa device that measures such reactions. Suitable devices include thosedisclosed in U.S. Application No. 62/244,188 filed Oct. 21, 2015, titled“Bed Bugs Detection Device,” and the U.S. non-provisional applicationand international PCT application thereof filed Oct. 21, 2016, which areincorporated by reference herein in their entireties, as well as U.S.Pat. Nos. 7,220,597 and 7,214,542, both of which are incorporated byreference herein in their entireties. In certain embodiments, thedetecting comprises performing a lateral flow assay. In certainembodiments, the detecting occurs in 1-20 minutes, 1-15 minutes, 1-10minutes, 1-5 minutes, 20 minutes or less, 15 minutes or less, 10 minutesor less, 5 minutes or less, within 20 minutes, within 15 minutes, within10 minutes, or within 5 minutes.

The amount of the antibody, antigen-binding fragment, mutant, conjugatedantibody or antigen-binding fragment, composition of the invention, or acombination thereof can include any effective amount, which will beapparent to a skilled practitioner based on known detection methods andthe methods disclosed in the examples. The sample can be diluted orundiluted.

In certain embodiments, the method further comprises collecting a samplecomprising the bed bug antigen with a collection device and extractingantigens from the sample. The sample can be collected from any surfaceassociated with bed bug infestation, including, but not limited to,bedding, mattresses, upholstery, carpets, rugs, and furniture. Thecollection device can be any suitable device, including but not limitedto a swab, such as a cotton swab, a vacuum, or any material that can beused to collect residue including, but not limited to, a wipe, tissue,or towelette. In certain embodiments the collection device is a swab. Incertain embodiments, extracting antigens from the sample comprisessolubilizing antigens in the sample with an extraction buffer. Suitableextraction buffers will be apparent to the skilled practitioner in viewof well-known extraction buffers and those disclosed in the examples.

EXAMPLES

The examples in dais section are offered by way of illustration, and notby way limitation.

Example 1 Generation of Anti-Bed Bug Monoclonal Antibodies

Mice were immunized with whole bed bug lysates and bed bug paperextracts (i.e., extracts from bed bug collection paper containing wastematerial from a bed bug colony).

Whole bed butt lysates were produced from nymphs, males, and femalesfrom a bed bug colony (Harlan strain, i2L,Research USA Inc., Baltimore,Md., USA) that were frozen and triturated in IX phosphate bufferedsaline (PBS). The lysates were clarified by 0.45 micron syringe filter.Protein concentration was determined by a standard Bradford proteinassay. The clarified, quantified extracts were aliquoted into 1.5 mLEppendorf tubes and stored at −80° C.

Bed bug collection paper (i2I,Research USA Inc., Baltimore, Md., USA)was cut into approximately 1 cm² pieces and placed into 2 mL plasticcentrifuge tubes. Extraction was performed by adding 1.0 mL of 50 mM PBS(pH 7.4) and mixing the tubes for 30 minutes on a tube rocker. After 30minutes, the extract was fully extracted by passing the mixture througha 5 mL syringe. This collected extract was then used to obtain moreextract from fresh collection paper by serially adding the extract tothe newly cut paper and repeating the process. The final extract wasthen centrifuged at 12,000 rpm for 10 minutes to remove particulates.The supernatant. was removed and retained, and the pelleted material wasdiscarded,. The protein concentration of the supernatant was determinedby Bradford assay to be 0.6 mg/mL. The final solution (i.e., “bed bugpaper extract”) was stored at −20° C.

Four 4-5 week old Barbie mice (Harlan Laboratories, Inc., Indianapolis,Ind., USA) were immunized subcutaneously in the back with 50 μg wholebed bug lysate mixed with 100 μl of adjuvant. The adjuvant used for twoof the mice was a traditional adjuvant (Freund's Adjuvant, Siama-AldrichCo. LLC, St. Louis., Mo., USA) and the adjuvant used for the remainingtwo mice was a water-soluble adjuvant (ImmuQuik®, KCH Scientific, SanJose, Calif., USA).

At Day 14 after the initial immunization, the immunized mice wereboosted with 50 μg whole bed bug lysate mixed with 100 μl of an adjuvantas originally used for each mouse. At Day 37, sera from the mice weretiter-tested using a standard enzyme immunoassay, using goat anti-mousehorseradish peroxidase conjugated antibody as the secondaryantibody/enzyme conjugate and 3,3′,5,5′-Tetramethylbenzidine as thechromogenic substrate, and 10 μg/ml whole bed bug lysate as the sourceof antigen. The two mice immunized with whole bed bug lysate in thewater-soluble adjuvant produced higher titers. However, since the titerswere not strong overall, all four immunized mice were boosted withdouble the amount of Whole bed bug lysate (i.e., 100 μg) at Day 51 andthen again at Day 78. At Day 107, the four mice were boosted with 15 μlof bed bug paper extract. At Day 117, the mouse with the highestprevious titer was titer-tested using whole bed bug lysate or bed bugpaper extract as the source of antigen. A weak reaction was observed forbed bug paper extract. At Day 134, all four mice were boosted with 100μl of bed bug paper extract using ImmuQuik® as the adjuvant. At Day 151,the mice were titer tested using bed bug paper extract as the source ofantigen, and the highest titer mouse was boosted with 100 μl bed bugpaper extract.

Spleen cells were collected from the two highest titer mice, one at Day154 and the other at Day 216, and fused with murine SP 2/0 myeloma cellsby using polyethylene glycol. The fused cells were cultured in selectionmedium for 10 days, followed by screening with bed bug paper extract asthe source of antigen. About 41 positive clones were identified fromprimary screening, and about 25 positive clones were confirmed insecondary screening. Stable cell lines were subcloned, ascites wereproduced for more than 25 clones, and antibodies were purified (e.g., inamounts of 2-5 mg). Two antibodies referred to herein as the BB2 and BB7antibodies were determined by enzyme immunoassay to have a strongreaction with bed bug paper extract as compared to antibodies from otherclones and were selected for further study. Hybridomas producing the BB2and. BB7 antibodies were deposited under the Budapest Treaty at theAmerican Type Culture Collection, Patent Depository, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, on Oct. 8, 2015, and givenATL″.(Accession No. PTA-122644 and ATCC Accession. No. PTA-122645,respectively.

Example 2 Sandwich Capture Assay of Anti-Bed Bug Monoclonal Antibodies

A sandwich capture assay was performed using the BB2 or BB7 antibody asa capture antibody and either gold-conjugated BB2 or gold-conjugated BB7as a detector antibody. Bed bug paper extract as described in Example 1was used as the source of bed bug antigen. A rabbit polyclonal anti-bedbug antibody as described in U.S. Publication No. 2015-0064727 was usedas a positive control capture antibody and PBS was used as a negativecontrol for the antigen.

Capture antibodies at concentrations of 2.0 mg/ml were spotted as 0.3 μldots onto nitrocellulose paper strips. Bed bug paper extract was addedto test strips and PBS was added to negative control strips.Gold-conjugated BB2 antibody (at pH 7 or 9) or gold conjugated BB7antibody (at pH 9) was added as the detector antibody to the strips asshown in FIG. 1. The negative control strips showed an absence ofbinding by detector antibodies. Test strips showed red staining of thecapture dots that indicated binding of the gold-conjugated detectorantibodies to bed bug antigen immobilized on the nitrocellulose by thecapture antibodies. As summarized in Table 1, strong reactions wereobserved with use of either the BB2 or BB7 antibody as the captureantibody. In contrast, very weak (i.e., “+”) or uncertain (i.e., “+/−”)reactions were observed with use of the rabbit polyclonal anti-bed bugantibody as the capture antibody.

TABLE 1 Observed Intensities in Sandwich Capture Assay Gold- Captureantibody conjugated Rabbit detector polyclonal antibody anti-bed bug(pH) antibody BB2 BB7 BB2 + ++++ +++ (7) BB2 + +++ ++ (9) BB7 +/− +++++++ (9)

Example 3 Lateral Flow Immunoassay to Detect Bed Bug Antigens

An example lateral flow immunoassay was designed to detect bed bugantigens from samples taken by swabbing areas of differing levels of bedbug Infestation, Extraction buffers were prepared and tested forefficient extraction of bed bug antigen from swabs, proper flow onnitrocellulose test strips, and low to no non-specific binding. Swabsamples were extracted and serially diluted to investigate thesensitivity of the assay. Precision of the assay Vias investigated bytesting replicates and reading signal intensities using a test stripreader.

Nitrocellulose membrane preparation: Nitrocellulose membranes (CN 140,25 mm, Sartorius Corp., Bohemia, N.Y., USA) were sprayed with 1.0 mg/ mLof the BB7 anti-bed bug antibody as the test line and 0.5 mg/mL of goatanti-mouse antibody (Lampire Biological Laboratories, Pipersville, Pa.,USA) as the control line using a Biodot Air Jet (Biodot. Irvine, Calif.,USA) for striping the nitrocellulose membranes. Striping Buffer was 1×PBS, 0.2% Sucrose, pH 7.4. The test line and control line were sprayed 7mm apart, with the test line located 10 mm from the bottom of themembrane. Membranes were striped at a rate of 1.0 μl/cm. The membraneswere dried at 37° C. for 1 hour and stored in a desiccated foil pouch.Striped membranes were kept desiccated overnight before blocking.

Nitrocellulose membrane blocking: After drying overnight, stripedmembranes were placed into a blocking solution (25 mM KPO₄, 0.2% Casein,0.5% Boric Acid, 0.02% Sucrose, 0.1% Surfactant 10G, 0.5% PVA) with theorientation of the test line at the bottom of the nitrocellulose and thecontrol line on the top of the nitrocellulose. The blocking solution wasallowed to wick to the top of the membrane. The membranes were removedfrom the blocking solution and placed in a finger rack to dry at 37° C.for 1 hour. Blocked membranes were kept desiccated in a plastic bag andstored in a dry room.

Antibody gold conjugation: A Slide-A-Lyzer™ disalysis cassette (10000molecular weight cutoff, Thermo Fisher Scientific Inc., Carlsbad,Calif., USA) was used to dialyze the BB2 anti-bed bug antibody overnightin 10 mM KPO₄, pH 7.4. The final concentration of the BB2 antibody afterdialyzing was 0.875 mg/ml. A colloidal gold solution containing 40 nmparticles and an optical density (OD) of 2:28 at 525 nm was adjusted atroom temperature to pH 8.6 with freshly made 0.1 M K₂CO₃. The dialyzedBB2 antibody was added to the colloidal gold solution while vortexing.The solution was incubated for 30 minutes on a rotator at roomtemperature. The conjugate was blocked with 10 μl (for every 1 ml of OD2 colloidal gold) of gold conjugate blocking buffer (25 mM KP04, 0.2%Bioterge, 6% BSA, 0,3% Sucrose) on a rotator at room temperature for 10minutes. The gold conjugate was centrifuged at 12000 RPM 4° C. for 20minutes and the supernatant was discarded. The conjugate pellet wasre-suspended with 0.2 ml (for every 1 ml of OD 2 colloidal gold)re-suspension buffer (1:5 dilution of gold conjugate blocking buffer in25 mM KPO₄, 0.05% Sodium Azide). The OD of the gold conjugated BB2antibody was checked using a spectrophotometer and adjusted to 10. Thegold conjugated BB2 antibody was stored at 4° C.

Gold conjugate pad preparation: A P-1000 pipette was used to saturate300 mm Ahlstrom 8950 glass fiber conjugate pads (Ahlstrom, Helsinki,Finland) with blocking buffer (25 mM KPO₄, 0.2% Casein, 0.5% Boric Acid,0.02% Sucrose, 0.1% Surfactant 10G, 0.5% MIA). After 15 minutes, thesaturated conjugate pads were transferred to a paper towel for a minute.Then, the conjugate pads were placed on a finger rack to dry at 37° C.for 1 hour. Blocked conjugate pads were put in a plastic bag withdesiccators and stored in a dry room. The OD10 gold-conjugated BB2antibody was prepared by adding 10% Sucrose and 5% Trehalose to theconjugate. The gold-conjugated antibody was dispensed onto the conjugatepads by an automatic striper (Matrix 160, Kinematic Automation, Inc.,Twain Harte, Calif., USA) at a dispensing rate of 10 μl/cm. Theconjugate pads were dried at 37° C. for 1 hour, packed in a desiccatedfoil pouch, and stored in a dry room.

Test strip lamination and cutting: The nitrocellulose membrane stripedwith the test antibody and control goat anti-mouse antibody waslaminated onto a vinyl backing card (G&L. Precision Die Cutting, SanJose, Calif., USA). A wick pad (30250, EMI Specialty Papers, Redding,Conn., USA) was placed on the top portion of the backing, overlappingthe membrane by 2 mm. A 10 mm conjugate pad was overlapped onto themembrane by 2 mm. A sample pad (Surewick 0048 cellulose pad, Millipore,Darmstadt, Germany) was placed on top of the conjugate pad with a 15 mmoverlap from the bottom of the backing card. Assembled cards were cutinto 4 mm strips using a cutter (CM4000, Biodot, Irvine, Calif., USA).

Extraction of test swabs: Swab samples were obtained from test siteshaving different levels of bed bug infestations, designated as levels of0, 2, 3, 4, 5, 7, and 8, with level 0 having the lowest level (i.e., nobed bugs) and 8 the highest level. Swabs were extracted in 350 μl ofextraction buffer for 1 minutes at room temperature in an Eppendorftube. Three extraction buffers were tested: extraction buffer 1contained 1× Tris-HCl (pH 7.6), 0.05% NaN₃, 0.1% BSA, and 0.1% Tween-20;extraction buffer 2 contained 1× Tris-HCl (pH 7.6), 0.05% NaN₃, 0.1%BSA, and 0.2% Tween-20; and extraction buffer 3 contained 1× Tris-HCl(pH 7.6), 0.05% NaN₃, 0.25% BSA, and 0.1% Tween-20, Serial dilutions ofthe swab extracts were performed from ½ to 1/4096.

Assay testing method: 70 μl of extraction buffer (negative control) orbed bug swab sample in extraction buffer was pipeted onto the samplepad. The test line intensity was read at 15 minutes by eye.

Extraction buffer 1 results; As shown in FIGS. 2-7, all strips showedpositive control lines for the binding of the goat anti-mouse antibodyto the gold-conjugated BB2 antibody. All strips showed the absence oftest lines for the negative control strip in which extraction buffer wasadded instead of a swab sample. FIG. 2 only shows positive control linesfor the level 0 swab sample dilutions since bed bug antigen was notpresent. In FIGS. 3-7, the positive control lines are the top lines, andany test lines showing the presence of bed bug antigen are beneath thepositive control lines. Dirt or insoluble particles from the slAabs werepresent near the bottom of the membranes for level 3, 5, 7, and 5 teststrips. The level 2 swab sample (FIG. 3) had visible test lines from the1/16 dilution to the ½ dilution. The level 3 swab sample (FIG. 4) hadvisible test lines from the 1/128 dilution to the ½ dilution. The level4 swab sample (FIG. 5) had visible test lines from the 1/1024 dilutionto the ½ dilution. The level 7 swab sample (FIG. 6) had visible testlines from the 1/4096 dilution to the ½ dilution. The level 8 swabsample (FIG. 7) had visible test lines from the 1/2048 dilution to the ½dilution. Although visually observed, some of the noted test lines arenot readily apparent for certain dilutions in FIGS. 3-7 due tophotographic limitations. All test lines were without smears. The signalintensity of the ½ dilution from the level 2 swab was approximatelyequal to that of the 1/32 dilution from the level 3 swab, the 1/64dilution front the level 4 swab, the 1/1024 dilution from the level 7swab, and the 1/512 dilution from the level 8 swab. Therefore, visiblesignal intensity of the ½ dilution from the level 2 swab was weakest,compared to that of the ½ dilutions from the level 3, 4, 7 and 8 swabs.The signal of the ½ dilution from the level 8 swab was the strongest.

Signal intensities were also determined using an Axxin test strip reader(Axxin, Fairfield, Victoria, Australia) to measure the test line areasfor different concentrations ( 1/2048 to ½) of level 0, 2, 3, 4, 7, and8 swab samples extracted in 350 μl of buffer 1. The results are shown inTable 2.

TABLE 2 Test Line Areas from Different Concentrations of Swab Samples(Buffer 1) Test Line Areas Concentration Level 8 Level 7 Level 4 Level 3Level 2 Level 0 0 253 397 232 267 237 362 1/2048 654 1187 — — — — 1/10241013 1978 359 — — — 1/512 1936 3291 443 336 297 — 1/256 3388 5356 681526 284 — 1/128 5373 7348 990 526 285 — 1/64 7662 10522 840 743 326 3271/32 9341 11385 3024 1199 310 296 1/16 11420 12550 4609 2095 411 265 ⅛12066 8830 7500 3309 392 433 ¼ 9793 7765 7980 6161 574 316 ½ 8693 62798550 8516 967 448

For each level, a reading was obtained for buffer as a negative control(i.e., concentration ‘0”). The negative control reading (Bo) was dividedby itself to yield a normalized value of 1. The negative control reading(Bo) was then divided by the test line area (B) for each dilution in thelevel, where smaller values under 1 suggest larger amounts of bed bugantigen and values above 1 it absence of bed bug antigen. The dataexpressed as Bo/B is provided in Table 3.

TABLE 3 Bo/B Calculated from Test Line Areas from DifferentConcentrations of Swab Samples (Buffer 1) Bo/B Concentration Level 8Level 7 Level 4 Level 3 Level 2 Level 0 0 1 1 1 1 1 1 [ 1/2048] 0.38690.3345 — — — — [ 1/1024] 0.2498 0.2007 0.6462 — — — [ 1/512] 0.13070.1206 0.5237 0.7946 0.798 — [ 1/256] 0.0747 0.0741 0.3407 0.5076 0.8345— [ 1/128] 0.0471 0.054 0.2343 0.5076 0.8316 — [ 1/64] 0.033 0.03770.2762 0.3594 0.727 1.107 [ 1/32] 0.0271 0.0349 0.0767 0.2227 0.76451.223 [ 1/16] 0.0222 0.0316 0.0503 0.1274 0.5766 1.366 [⅛] 0.021 0.0450.0309 0.0807 0.6046 0.836 [¼] 0.0258 0.0511 0.0291 0.0433 0.4129 1.146[½] 0.0291 0.0632 0.0271 0.0314 0.2451 0.808

Tables 2-3 and FIG. 8A show, in general, that the measured values ofconcentrations from level 8 swabs (corresponding to the greatest levelof bed bug infestation) were highest and values of concentrations fromlevel 2 swabs (corresponding to the smallest level of bed buginfestation) were lowest. Table 2 and FIG. 8B show that level 8 swabsproduced better signal intensities than level 2 and level 4 swabs. Table3 suggests that level 8 swabs contained more bed bug antigen than otherlevels.

Extraction buffer 2 results: As shown in FIGS. 9-11, all strips showedpositive control lines for the binding of the goat anti-mouse antibodyto the gold-conjugated BB2 antibody. All strips showed the absence oftest lines for the negative control strip in which extraction buffer wasadded instead of a swab sample, in FIGS. 9-11, the positive controllines are the top lines, and any test lines showing the presence of bedbug antigen are beneath the positive control lines. Dirt or insolubleparticles from the swabs were present near the bottom of the membranesfor level 4, 5, and 8 test strips The level 4 swab sample (FIG. 9) hadvisible test lines from the 1/512 dilution to the ½ dilution. The level5 and 8 swab samples (FIGS. 10 and 11, respectively) had visible testlines from the 1/1024 dilution to the ½ dilution. Although visuallyobserved, some of the noted test lines are not readily apparent forcertain dilutions in FIGS. 9-11 due to photographic limitations. Alltest lines were smeared. The signal intensity of the 1/16 dilution fromthe level 4 swab was approximately equal to that of the 1/64 dilutionfrom the level 5 and 8 swabs. Signal intensity was weakest for the level4 swab and similar between the level 5 and 8 swabs.

Signal intensities were also determined using an Axxin test strip reader(Axxin, Fairfield, Victoria, Australia) to measure the test line areasfor different concentrations ( 1/2048 to ½) of level 4, 5, and 8 swabsamples extracted in 350 μl of buffer 2. The results are shown in Table4.

TABLE 4 Test Line Areas from Different Concentrations of Swab Samples(Buffer 2) Test Line Areas Concentration Level 8 Level 5 Level 4 0 195230 217 1/2048 365 — — 1/1024 693 542 356 1/512 961 810 446 1/256 17871395 527 1/128 3250 2143 959 1/64 5225 4157 1623 1/32 7927 6696 26921/16 9306 9766 4413 ⅛ 10647 10173 6685 ¼ 11489 — 8605 ½ 9818 12250 10150

The data expressed as Bo/B calculated from test line areas is providedin Table 5.

TABLE 5 Bo/B Calculated from Test Line Areas from DifferentConcentrations of Swab Samples (Buffer 2) Bo/B Concentration Level 8Level 5 Level 4 0 1 1 1 [ 1/2048] 0.5342 — — [ 1/1024] 0.2814 0.42440.6096 [ 1/512] 0.2029 0.2840 0.4865 [ 1/256] 0.1091 0.1649 0.4118 [1/128] 0.06 0.1073 0.2263 [ 1/64] 0.0373 0.0553 0.1337 [ 1/32] 0.02460.0343 0.0806 [ 1/16] 0.0210 0.0236 0.0492 [⅛] 0.0183 0.0226 0.0325 [¼]0.0170 — 0.0252 [½] 0.0199 0.0188 0.0214

Tables 4-5 and FIG. 12A show that, in general, measured values of level8 swabs were the highest and values of level 4 swabs were the lowest.Although visibility of test lines from level 5 swabs were similar tolevel 8 swabs (FIGS. 10 and 11, respectively), Axxin reader results inTable 4 and FIG. 12A show that level 8 swabs produced better signalintensities than level 4 and 5 swabs. Table 5 suggests that level 8swabs contained more bed bug antigen than the other levels.

Extraction buffer 3 results: As shown in FIGS. 13-17, all strips showedpositive control lines for the binding of the goat anti-mouse antibodyto the gold-conjugated BB2 antibody. All strips showed the absence oftest lines for the negative control strip in which extraction buffer wasadded instead of a swab sample. In FIGS. 13-17, the positive controllines are the top lines, and any test lines showing the presence of bedbug antigen are beneath the positive control lines. Dirt or insolubleparticles from the swabs were present near the bottom of the membranesfor level 3, 5, 7, and 8 test strips. The level 2 swab sample (notshown) had visible test lines from the ⅛ dilution to the ½ dilution. Thelevel 3 swab sample (FIG. 13) had visible test lines from the 1/128dilution to the ½ dilution. The level 4 swab sample (FIG. 14) hadvisible test lines from the 1/1024 dilution to the ½ dilution. The level5 swab sample (FIG. 15) had visible test lines from the 1/512 dilutionto the ½ dilution. The level 7 swab sample (FIG. 16) had visible testlines from the 1/4096 dilution to the ½ dilution. The level 8 swabsample (FIG. 17) had visible test lines from the 1/2048 dilution to the½ dilution. Although visually observed, some of the noted test lines arenot readily apparent for certain dilutions in FIGS. 13-17 due tophotographic limitations. All test lines were smeared. The signalintensity of the ½ dilution from the level 2 swab was approximatelyequal to that of the 1/64 dilution from the level 3 swab, the 1/64dilution from the level 4 swab, the 1/64 dilution from the level 5 swab,the 1/1024 dilution from the level 7 swab, and the 1/512 dilution fromthe level 8 swab. Therefore, visible signal intensity of the ½ dilutionfrom the level 2 swab was weakest compared to that of the ½ dilutionsfrom the other levels. The signal of the ½ dilution from the level 8swab was the strongest.

Signal intensities were also determined using an Axxin test strip reader(Axxin, Fairfield, Victoria, Australia) to measure the test line areasfor different concentrations ( 1/2048 to ½) of level 4, 5, and 8 swabsamples extracted in 350 μl of butler 3. The results are shown in Table6.

TABLE 6 Test Line Areas from Different Concentrations of Swab Samples(Buffer 3) Test Line Areas Concentration Level 8 Level 7 Level 5 Level 4Level 3 Level 2 0 441 283 263 225 170 231 1/2048 735 781 — — — — 1/1024798 1037 270 334 — — 1/512 1414 2003 385 574 309 261 1/256 2691 2998 4221090 403 261 1/128 4741 4665 1026 1439 522 285 1/64 7173 6912 2013 2403724 276 1/32 9603 8746 5763 4090 1360 269 1/16 10377 9626 5743 5842 2430271 ⅛ 11788 11767 7021 8061 3462 340 ¼ 10725 8466 7120 9518 5189 406 ½8584 6803 9408 10413 6098 752

The data expressed as Bo/B calculated from test line areas is providedin Table 7.

TABLE 7 Bo/B Calculated from Test Line Areas from DifferentConcentrations of Swab Samples (Buffer 3) Bo/B Concentration Level 8Level 7 Level 5 Level 4 Level 3 Level 2 0 1 1 1 1 1 1 [ 1/2048] 0.60.3624 — — — — [ 1/1024] 0.5526 0.2729 0.9741 0.6737 — — [ 1/512] 0.31190.1413 0.6831 0.3920 0.5502 0.8851 [ 1/256] 0.1639 0.0944 0.6232 0.20640.4218 0.8851 [ 1/128] 0.0930 0.0607 0.2563 0.1564 0.3257 0.8105 [ 1/64]0.0615 0.0409 0.1307 0.0936 0.2348 0.8370 [ 1/32] 0.0459 0.0324 0.04560.0550 0.1250 0.8587 [ 1/16] 0.0425 0.0294 0.0458 0.0385 0.0700 0.8524[⅛] 0.0374 0.0241 0.0375 0.0279 0.0491 0.6794 [¼] 0.0411 0.0334 0.03690.0236 0.0328 0.5690 [½] 0.0514 0.0416 0.0280 0.0216 0.0279 0.3072

Tables 6-7 and FIG. 18A show that, in general, measured values of level8 swabs were highest and values of level 2 swabs were lowest. Table 7and FIG. 18A show that level 8 swabs produced better signal intensitiesthan the other levels.

Comparison of extraction buffers: For extraction buffer 1. all testlines were clear and did not have smears, while extraction buffers 2 and3 resulted in smeared test lines. For level 4 swabs, extraction withbuffers 1 and 3 resulted in signals starting at the 1/1024 dilution, butextraction with buffer 2 resulted in signals starting at the 1/512dilution. However, extraction of level 5 with buffer 2 yielded signal ata lower concentration ( 1/1024 dilution) than extraction with buffer 3 (1/512 dilution). Buffers I and 3 yielded signals at the sameconcentration for level 3 and 7 swabs. However, extraction of level 2swabs with buffer 1 resulted in a signal at, a lower concentration (¹/₁6dilution) than extraction with buffer 3 (⅛ dilution). In generalmeasured values of all test level swabs were higher for extraction withbuffer 1 than for buffers 2 and 3.

Precision study using Axxin reader: Nine replicates of extraction buffer1 as a negative control and nine replicates each of 1/2048, 1/512, and1/128 dilutions of level 7 swabs extracted with buffer 1 were preparedand test line areas were measured using an Axxin test strip reader(Axxin, Fairfield, Victoria, Australia). Test line areas are shown inTable 8 and FIG. 20.

TABLE 8 Precision Study Results of Replicate Test Line Areas Test LineAreas Level 7 Level 7 Level 7 Trial Buffer 1 1/2048 1/512 1/128 1 2501415 3299 6163 2 251 1426 2794 6276 3 233 1293 2684 7305 4 289 1238 28108079 5 260 1202 2531 8090 6 271 1075 2401 6583 7 260 1283 2260 9109 8218 1540 2660 8297 9 194 1444 2773 5997 Average 247.3 1324.0 2690.27322.1 STDEV 28.6 144.3 295.3 1120.6 % CV 11.6 10.9 11.0 15.3

The percent coefficient of variation (% CV) was less than 20%, which isa good % CV for Axxin measurements.

The invention is not to be limited in scope by the specific embodimentsdescribed herein, indeed, various modifications of the invention inaddition to those described will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the, appendedclaims.

All references (e.g., publications or patents or patent applications)cited herein are incorporated herein by reference in their entirety andfor all purposes to the same extent as if each individual reference(e.g., publication or patent or patent application) was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

Other embodiments are within the following claims.

1. An antibody produced by a hybridoma deposited at the American TypeCulture Collection (ATCC) under Accession Number PTA-122644 [BB2] orPTA-122645 [BB7], or an antigen-binding fragment thereof.
 2. (canceled)3. A monoclonal antibody or an antigen-binding fragment thereofcomprising the heavy and light chain complementarity determining regionsof the antibody of claim
 1. 4. The antibody or antigen-binding fragmentof claim 3, comprising the heavy and light chain variable regions of theantibody of claim
 1. 5. The antibody or antigen-binding fragment ofclaim 4, comprising the heavy and light chains of the antibody ofclaim
 1. 6. The antibody or antigen-binding fragment of claim 1, whereinthe antibody or antigen-binding fragment is capable of binding to a bedbug antigen in a lysate of whole bed bugs or an extract of collectionpaper comprising bed bug waste material.
 7. A mutant of the antibody orantigen-binding fragment of claim 1, wherein the mutant is capable ofbinding to a bed bug antigen in a lysate of whole bed hugs or an extractof collection paper comprising bed bug waste material.
 8. (canceled) 9.A conjugated monoclonal antibody or conjugated antigen-binding fragment,comprising the antibody or antigen-binding fragment of claim 1, and adetection agent.
 10. The conjugated antibody or conjugatedantigen-binding fragment of claim 9, wherein the detection agent iscolloidal gold.
 11. The conjugated antibody or conjugatedantigen-binding fragment of claim 9, comprising the antibody produced bythe hybridoma deposited at the ATCC under Accession Number PTA-122644[BB2], or an antigen binding fragment thereof.
 12. The conjugatedantibody or conjugated antigen-binding fragment of claim 9, comprisingthe antibody produced by the hybridoma deposited at the ATCC underAccession Number PTA-122645 [BB7], or an antigen binding fragmentthereof.
 13. A composition comprising the antibody or antigen-bindingfragment of claim
 1. 14. A composition comprising: (a) an antibodyproduced by a hybridoma deposited at the American Type CultureCollection (ATCC) under Accession Number PTA-122644 [BB2], and (b) anantibody produced by a hybridoma deposited at the American Type CultureCollection (ATCC) under Accession Number PTA-122645 [BB7], or theconjugated antibody of claim
 12. 15. A composition comprising: (a) anantibody produced by a hybridoma deposited at the American Type CultureCollection (ATCC) under Accession Number PTA-122645 [BB7], and (b) anantibody produced by a hybridoma deposited at the American Type CultureCollection (ATCC) under Accession Number PTA-122644 [BB2], or theconjugated antibody of claim
 11. 16. A kit comprising the antibody orantigen-binding fragment of claim
 1. 17. A hybridoma capable ofproducing an antibody, wherein the hybridoma is deposited at the ATCCunder Accession Number PTA-122644 [BB2] or PTA-122645 [BB7]. 18.(canceled)
 19. An isolated cell producing the antibody orantigen-binding fragment of claim
 1. 20. A method of making the antibodyor antigen-binding fragment of claim 1, comprising (a) culturing a cellthat produces the antibody or antigen-binding fragment, and (b)isolating the antibody or antigen-binding fragment from the culturedcell.
 21. A method of detecting bed bugs, comprising contacting a samplecomprising a bed bug antigen with the antibody or antigen-bindingfragment of claim 1, and detecting binding of the bed bug antigen to theantibody or antigen-binding fragment.
 22. (canceled)
 23. A method ofdetecting bed bugs comprising contacting a sample comprising a bed bugantigen with an antibody produced by a hybridoma deposited at theAmerican Type Culture Collection (ATCC) under Accession NumberPTA-122644 [BB2], and conjugated antibody of claim
 12. 24. A method ofdetecting bed bugs, comprising contacting a sample comprising a bed bugantigen with an antibody produced by a hybridoma deposited at theAmerican Type Culture Collection (ATCC) under Accession NumberPTA-122645 [BB7], and conjugated antibody of claim
 11. 25. The method ofclaim 21, wherein the detecting comprises performing a lateral flowassay.
 26. The method of claim 21, wherein the method further comprisescollecting the sample comprising the bed hug antigen with a collectiondevice and extracting antigens from the sample.
 27. The method of claim26, wherein the collection device is a swab.